Glycan Microarray Analysis of Lectin Binding

A glycan microarray analysis was performed by Ebiogen (Seoul, Korea) according to previous methods [23 (link)]. The Glycan Array kit was purchased from RayBioTech (Norcross, GA, USA). An array containing 300 synthetic glycans printed in quadruplicate on a glass slide was used. Label-based detection was performed according to the manufacturer’s protocols and previous methods [23 (link)]. Biotinylated recombinant lectins and native lectins at 50 μg/mL were added to the array wells and incubated for >3 h with gentle rocking. The glass slide was washed with 1× wash buffer I and II, provided in the kit. Glycan-lectin binding was detected by incubation with Cy3 equivalent dye-conjugated streptavidin for 1 h at room temperature. For cyanine-3 detection, the signals were visualized using a microarray laser scanner (GenePix 4100A; Molecular Devices, Sunnyvale, CA, USA) with excitation at 554 nm and emission at 568 nm. Data extraction was performed using the microarray analysis software, GenePix Pro (ver. 7.2, Sunnyvale, CA, USA). Glycan array data were normalized and analyzed using RayBio Analysis software (RayBioTech, GA-Glycan-300-SW, Norcross, GA, USA).